B and T cells express receptors that recognize an almost infinite variety of antigens. The diversity of these receptors is generated through a process known as V(D)J recombination. This involves repeated cleavage and repair of antigen receptor genes, which are scattered across several different chromosomes. Recombination is carefully regulated and occurs at different chromosomal loci in a specific sequence. This normally proceeds without mishap, but mistakes do sometimes occur. Deficiencies in components of the nonhomologous end joining (NHEJ) and DNA damage response pathways are known to predispose to aberrant V(D)J rearrangement events that can lead to leukemias and lymphomas. In large part, the spectrum of damage will depend on the contribution of each factor to the repair of RAG-mediated double strand breaks. The effects of individual repair and damage response factors in this context have been extensively studied, but beyond this, little is known about whether any of these factors have a role in regulating RAG enzymatic activity or the accessibility of the antigen receptor loci and surrounding genes to halt cleavage after RAG-mediated breaks are introduced on one allele. The work outlined in this grant will lead to a more detailed and mechanistic understanding of how programmed DNA breaks are introduced in a controlled manner to protect genome stability during V(D)J recombination. We aim to look at the 3D organization of the genome in developing T cells to identify 'at risk' candidates that are closely associated with Tcra/d in cells where they are being recombined. Further, we will determine whether intimate relationships are fostered by looping out of actively transcribed genes and congregation in transcription factories that could be enriched for RAG proteins. The combined analyses of nuclear organization and gene activity (which is known to be an important determinant of RAG targeting) will provide us with a starting point from which to unravel the effects of individual repair factors. Importantly, this analysis will allow us to put known oncogenc translocation events into a global context so that we can begin to understand why particular genes are targeted and join with Tcra/d in chromosomal rearrangements. )